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Dissertation
A colloidal science approach to the study of atherosclerosis
Doctor of Philosophy (Ph.D.), Drexel University
Jan 2006
DOI:
https://doi.org/10.17918/etd-874
Abstract
This work examines three related, but previously unexplored, aspects of membrane biophysics and colloid science in the context of atherosclerosis. First, it is shown that Sphingomyelinase (SMase)-induced aggregation of low density lipoproteins (LDL), coupled with LDL exposure to cholesterol esterase (CEase), results in nucleation of cholesterol crystals; long considered the hallmark of atherosclerosis. This is measured utilizing a time dependent Förester Resonance Energy Transfer (FRET) assay using the membrane probes ergosta-5,7,9(11),22-tetraen-3b-ol (DHE) and 1-acyl-2-[12-[(5-dimethylamino-1-naphthalenesulfonyl)amino]dodecanoyl]-sn-Glycero-3-phosphocholine (DL). In particular, this study reveals that cholesterol nucleation from LDL can be quantified and the order of enzyme addition does not affect the propensity of LDL to nucleate cholesterol crystals. This raises the possibility that nucleation can proceed from either the intra- or extra-cellular space. Second, using a combination of dynamic light scattering and UV/Vis absorbance spectroscopy to measure aggregation kinetics and particle sizes, a mass action model was developed to describe the aggregation process of LDL upon their exposure to SMase. It is found that LDL aggregation is independent of the relative concentrations of LDL and SMase, but rather depends on the LDL-to-SMase molar ratio. An important finding of this work was that the aggregate size was found to be a critical factor in foam cell formation as determined by an increase in cellular cholesterol content upon incubation with J774A.1 cells. Finally, the interactions between cholesterol (Chol) and sphingomyelin (SM) were investigated. Specifically, it is demonstrated that ceramide-rich aggregates of LDL release cholesterol to neighboring vesicles far more rapidly, and to a greater extent, than does native LDL. A likely explanation for this observation is due to the loss of the SM-Chol interaction and the displacement of cholesterol from SM-Chol rafts by "raft-loving" ceramide. Moreover, a time-independent FRET assay is used to measure SM-Chol raft sizes in model membrane systems. Taken together, these findings point to the possibility of an extracellular nucleation mechanism and underscore the important role that biological colloids play in human disease.
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Details
- Title
- A colloidal science approach to the study of atherosclerosis
- Creators
- Andrew Joseph Guarino - DU
- Contributors
- Steven P. Wrenn (Advisor) - Drexel University (1970-)
- Awarding Institution
- Drexel University
- Degree Awarded
- Doctor of Philosophy (Ph.D.)
- Publisher
- Drexel University; Philadelphia, Pennsylvania
- Resource Type
- Dissertation
- Language
- English
- Academic Unit
- Chemical (and Biological) Engineering [Historical]; College of Engineering (1970-2026); Drexel University
- Other Identifier
- 874; 991014632388604721